Background: Copper (Cu) binds to amyloid beta (Aβ), generating reactive oxygen species (ROS) and altering cholesterol metabolism, leading to accumulation that worsens Alzheimer’s disease (AD). Cu near amyloid plaques can mediate inflammation via the NOD-like receptor protein 3 (NLRP3) inflammasome, which activates caspase-1 and interleukin-1β (IL-1β). Whether Cu exacerbates Aβ-induced inflammasome activation and microglial dysfunction remains unclear. Methods: Mouse microglia (SIM-A9) were treated with 1 µM Aβ and 100 µM Cu for 24 hours. ROS production was measured using dihydroethidium (DHE). Cholesterol levels were assessed via confocal microscopy and the Amplex Red kit. Inflammasome and phagocytosis proteins were analyzed by Western blot and PCR. Phagocytosis was measured using fluorescent microbeads, while inflammasome assembly was visualized with ASC-GFP speckle formation. Results: Cu increased IL-1β and NLRP3 expression, with a synergistic effect observed during Cu+Aβ treatment. Cu and Cu+Aβ elevated total and mitochondrial cholesterol levels while reducing glutathione (GSH). ROS production increased but was mitigated by glutathione ester (GSHee) and mitochondrial antioxidants (MitoTEMPO and MitoQ). Microglial phagocytosis, impaired by Cu and Cu+Aβ, was partially restored by GSHee. Cu also reduced phagocytosis proteins (ABCA7 and CD36) and caused their cytosolic redistribution, leading to decreased phagocytic activity. Conclusions: Elevated Cu levels exacerbate Aβ-induced microglial dysfunction by increasing mitochondrial oxidative stress, cholesterol accumulation—both at the cellular and mitochondrial level—and inflammasome activation. These effects impair phagocytosis, contributing to Aβ plaque accumulation and worsening AD pathology.

This research was funded by MCIN/AEI/10.13039/501100011033 and co-funded by NextGenera-tionEU/PRTR and “ERDF A way of making Europe”; grants PID2022-143279OB-100 and RED2022-134786-T, AGAUR, and grant 2021-SGR00490, respectively; and by the CIBERNED (Convocatoria 2022 Proyectos Colaborativos 2020/21).